By Tanuja Rohatgi, Checkbiotech
DPT vaccine, a combination of diphtheria, pertussis and tetanus
vaccines, is one of the many vaccines given to children to protect them from
life threatening bacterial infections. To eliminate the chances of rare, but
severe side-effects of DPT immunization, in addition to a reduced production
cost, Dr. Alpuche-Solis' team investigates a possible safer and more
efficient method.
With the advent of molecular biology in early 1980´s, scientists
worked on developing and producing vaccines from purified antigen - known as
subunit vaccines - instead of using whole inactivated or attenuated
microorganisms as vaccines. This minimizes the chances of side-effects
usually associated with whole microorganisms (that have been "killed") used
in conventional vaccines.

Although subunit vaccines are safe to produce and easy to use, they
too have their limitations. The conventional production of subunit vaccines
is cost- intensive as it requires large fermentors and refrigeration. Also,
most of the subunit vaccines are administered via injection and this brings
along the risk of disease transmission if syringe and needles are
contaminated, especially in the developing world where they are readily
needed.

Production of vaccines in plants is an attractive alternative to
overcome several problems faced in fermention-based, subunit vaccine
production. Plants can be genetically modified to produce edible vaccines
capable of generating protective immune response with high efficacy.
Plant-based vaccines are safe to use and inexpensive to produce.

Towards this aim, Dr. Alpuche-Solis and his colleagues at the Potosino
Institute for Science and Technology Investigation (Instituto Potosino de
Investigación Científica y Tecnológica) in Mexico set out to generate
transgenic tomato plants that would express DPT antigenic peptides. Such a
tomato has the potential of being used as an edible, multi-component DPT
vaccine, which can improve safety and reduce production costs.

DPT tomato preparation

To generate such a transgenic plant, Dr. Alpuche knew there were some
important steps to consider. The first step was to modify the bacterial
genes that code for the diphtheria, pertussis and tetanus toxins to better
production rate in tomato using synthetic genes optimized for this
vegetable.

Then, additional modifications were made to ensure that the plants
would produce large amounts of the desired proteins, while also ensuing that
the proteins would be retained in the correct part of the tomato cells.

After the needed preparation, the researchers then inserted the
genetic sequence into the tomato plant's genome. As the plants grew, Dr.
Alpuche-Solis' team noticed that the transgenic plants were healthy and
showed no visible defects.

Once the tomatoes were large enough, they then performed various tests
to estimate how much protein they had both in leaves and fruit and to test
whether or not the activity of DPT polypeptide from the tomato plants would
still be effective for vaccine use.

When contacted by Checkbiotech, Dr. Alpuche-Solis explained, "In the
best transgenic line obtained, approximately 270 mg of freeze-dried tomato
had concentrations of diphtheria and tetanus antigens, similar to the
concentrations found in the commercial vaccine Quadracel."

Dr. Alpuche-Solis further added that, "Multiple dosages in mice tests
could help increase the antibodies response to the pertussis antigen."

When asked why tomatoes were chosen, Dr. Alpuche-Solis responded, "We
chose tomatoes, because we have established a good protocol in our
laboratory for tomatoes, and because it is one of the most popular
vegetables in the world and very high yields can be obtained in greenhouse
production"

Dr. Alpuche-Solis also explained further advantages of the tomato,
such as it is consumed fresh without further processing and that it has a
short life cycle. When all these characteristics are taken into
consideration, Dr. Alpuche-Solis suggests the tomato is a promising model
for the edible vaccines.

Next stage: increase DPT

Dr. Alpuche-Solis also explained that his research group is pressing
forward with a new strategy that would increase the amount of DPT protein in
the plant. "As a first step we have used biolistic transformation in tobacco
plants with a gene optimized for chloroplast expression, and we have
regenerated tobacco plants and currently we are analyzing the expression
levels". Once success can be established in tobacco plants, then Dr.
Alpuche-Solis' research team can move onto tomatoes.

Checkbiotech has also learned that initial experiments, where the DPT
tomato plants were fed to mice have shown promising results. Elaborating on
the results, Dr. Alpuche-Solis noted, "We have already done some experiments
with mice, and they were immunized by oral route with the plant material in
three weekly doses. We found that the mice showed serological responses to
pertussis, tetanus and diphtheria antigens. We also analyzed the
tracheopulmonary fluid and gut washes and we found a humoral response."

These results are very promising because it means the DPT polypeptides
expressed in plants are functional and that they are capable of inducing
immune responses in vivo, which is a good sign that the vaccination may
work. The next step is to perform the challenge against the toxins to
demonstrate protection in mice.

Although producing vaccines in transgenic plants is an innovative
concept with significant benefits, there are genuine concerns about the
possibility of cross-pollination. In order to avoid mixing, the transgenic
tomatoes will be grown in greenhouses under supervision where the absence of
insects favors self-pollination and reduce out-crossing with wild relatives.
In addition, chloroplast transformation reduces gene flow due to the
maternal heredity; therefore this pollen does not contain the transgene.

Dr. Alpuche-Solis is a great believer in the concept of biodiversity
and genetic evolution. "Biodiversity is the base of evolution and gene flow
has happened naturally for years therefore it should not be a concern about
gene flow of a transgenic material."

Further he added, "FDA and EPA in America and counterparts in Europe
have demonstrated that transgenic crops are safe for humans and the
environment. There are more economic issues behind the concerns that
influenced public polices, however, each year the global surface of GM crops
continues to increase, reflecting the growing acceptance of this technology
by farmers of both industrialized countries and developing nations

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